ABSTRACT
OBJECTIVE To clone and express toxin C (tcdC) gene pathogenicity locus of Clostridium difficile (CD), and prepare the polyclonal antibody against TcdC protein. METHODS The tcdC gene was amplified from CD (ATCC43255) strain genome DNA and inserted into prokaryotic expression vectors. The correct recombinant plasmids were transformed into E.coli which was induced to express the GST-TcdC/L and IL1-TcdC/L fusion protein. Meanwhile, the fusion proteins were respectively purified through Ni-NTA agarose affinity chromatography and Q-Sepharose Fast Flow. The purified GST-TcdC/L fusion protein was used as an antigen to inoculate rabbits to produce antiserum. Two weeks after the final immunization, the rabbits were sacrificed and serum was collected. The titer of the serum was deter-mined by ELISA and the reactivity of the polyclonal antibody was identified by Western blotting. RESULTS The SDS-PAGE result showed that GST-TcdC/L and IL1-TcdC/L fusion proteins were expressed at the size of 45 ku and 39 ku. The titer of TcdC/L polyclonal antibody was over 1:6.4 × 104. Western blotting detection demonstrated that the TcdC/L polyclonal antibody recognized the TcdC protein in CD (ATCC43255). CONCLUSION The tcdC/L gene is cloned and the polyclonal antibody against TcdC/L is prepared, which will contribute to studing the function and mechanism of tcdC gene in pathogenicity locus.
ABSTRACT
Objective To investigate the alterations of tight junction proteins in duodenal mucosa from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease(PD) model. Methods Rats were subjected with 6-OHDA in bilateral substantia nigra (SN) as PD model. The location and quantitative detecting techniques inclu-ding immunohisto- chemistry and western blotting were used to determine the expressions and alterations of tight junction proteins (claudin-1, occludin, ZO-1). Results Claudin-1, occludin and ZO-1 were highly expressed in the duodenal mucosa of PD models, where the expressions of occluding (P<0.001) and ZO-1 (P<0.001) were much lower, while the expression of claudin-1 had no alteration. Conclusions The down-regulated expres-sion of occludin and ZO-1 was detected in duodenal mucosa of PD models, which might be related to duodenal ulcer in PD.
ABSTRACT
Objective To investigate the alterations of tight junction proteins in duodenal mucosa from 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease(PD) model. Methods Rats were subjected with 6-OHDA in bilateral substantia nigra (SN) as PD model. The location and quantitative detecting techniques inclu-ding immunohisto- chemistry and western blotting were used to determine the expressions and alterations of tight junction proteins (claudin-1, occludin, ZO-1). Results Claudin-1, occludin and ZO-1 were highly expressed in the duodenal mucosa of PD models, where the expressions of occluding (P<0.001) and ZO-1 (P<0.001) were much lower, while the expression of claudin-1 had no alteration. Conclusions The down-regulated expres-sion of occludin and ZO-1 was detected in duodenal mucosa of PD models, which might be related to duodenal ulcer in PD.